Successful re-ossification of calvarial defects is characteristically limited to immature animals and children less than 1-2 years of age. Conversely, skeletally mature animals demonstrate an almost universal inability to heal even small trephine defects, with bone deficits remaining present for the life of the subject. While a plethora of strategies have been developed over the past century for treating adult calvarial defects, the myriad of methods currently available reflects the inadequacies of each therapeutic technique. By combining advances in developmental biology, organogenesis, stem cell biology, bioengineering, and material sciences, however, a new paradigm for calvarial bone tissue engineering has emerged - Regenerative Medicine. Of the multitude of applications for tissue engineering and regenerative medicine, calvarial defects represent one of the most likely targets to meet with clinical success, given the alluring potential for implementation of translational therapies in the near future. This proposal seeks to significantly expand the scope of our parent grant proposal in determining the optimal design of a cell-based calvarial regenerative strategy utilizing human adipose-derived stromal cells (hASCs). In Specific Aim 1, we will investigate the capacity of concurrent controlled exposure of hASC to exogenous rhBMP-2 and RNAi-mediated suppression of BMP antagonism over a critical window of osteogenic differentiation to enhance in vitro osteogenesis relative to independent manipulations. In Specific Aim 2, we will assay the ability of similar manipulations of hASC to regenerate bone in vivo in our critical sized calvarial defect nude mouse model. We will employ the use of PEG-based hydrogel scaffolds to deliver the cells and determine if skeletal healing can be augmented by modulating BMP signaling via microparticle encapsulated recombinant human BMP-2 delivery and RNA interference of Noggin. We will also be able to examine the respective contributions of the implanted donor and surrounding host cells to the regenerate. Ultimately, the translational goal of this application is to realize a cell-based clinical regenerative medicine strategy to repair calvarial defects using tissue engineered bone. PUBLIC HEALTH RELEVANCE: Adult animals demonstrate an almost universal inability to heal even small skull defects, with bone deficits remaining present for the life of the subject. While many strategies have been developed over the past century for treating adult skull defects, the myriad of methods currently available reflects the inadequacy of each therapeutic technique. By combining advances in developmental biology, organogenesis, stem cell biology, bioengineering, and material sciences, however, a new paradigm for bone tissue engineering has emerged - Regenerative Medicine. Of the multitude of applications for tissue engineering and regenerative medicine, adult skull defects represent one of the most likely targets to meet with clinical success, given the alluring potential for implementation of translational therapies in the near future. This Competitive Revision proposal seeks to optimize the skull regenerative strategy utilizing human fat-derived multipotent cells outlined in our parent grant through simultaneous targeted molecular manipulations of pro-osteogenic agonists and antagonists.